2.1. Rhabdoviruses
Rhabdoviruses are membrane-enveloped viruses that are widely distributed in nature where they infect vertebrates, invertebrates, and plants. There are two distinct genera within the rhabdoviruses, the Lyssavirus genus and the Vesiculovirus genus. Rhabdoviruses have single, negative-strand RNA genomes of 11–12,000 nucleotides (Rose and Schubert, 1987, Rhabdovirus genomes and their products, in The Viruses: The Rhabdoviruses, Plenum Publishing Corp., NY, pp. 129–166). The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins, termed N (nucleocapsid), P (formerly termed NS, originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle. Because the genome is the negative sense [i.e., complementary to the RNA sequence (positive sense) that functions as mRNA to directly produce encoded protein], rhabdoviruses must encode and package an RNA-dependent RNA polymerase in the virion (Baltimore et al., 1970, Proc. Natl. Acad. Sci. USA 66: 572–576), composed of the P and L proteins. This enzyme transcribes genomic RNA to make subgenomic MRNAS encoding the 5–6 viral proteins and also replicates full-length positive and negative sense RNAs. The genes are transcribed sequentially, starting at the 3′ end of the genomes. The same basic genetic system is also employed by the paramyxoviruses and filoviruses.
The prototype rhabdovirus, vesicular stomatitis virus (VSV), grows to very high titers in most animal cells and can be prepared in large quantities. As a result, VSV has been widely used as a model system for studying the replication and assembly of enveloped RNA viruses. The study of VSV and related negative strand viruses has been limited by the inability to perform direct genetic manipulation of the virus using recombinant DNA technology. The difficulty in generating VSV from DNA is that neither the full-length genomic nor antigenomic RNAs are infectious. The minimal infectious unit is the genomic RNA tightly bound to 1,250 subunits of the nucleocapsid (N) protein (Thomas et al., 1985, J. Virol. 54:598–607) and smaller amounts of the two virally encoded polymerase subunits, L and P. To reconstitute infectious virus from the viral RNA, it is necessary first to assemble the N protein-RNA complex that serves as the template for transcription and replication by the VSV polymerase. Although smaller negative-strand RNA segments of the influenza virus genome can be packaged into nucleocapsids in vitro, and then rescued in influenza infected cells (Enami et al., 1990, Proc. Natl. Acad. Sci. USA 87:3802–3805; Luytjes et al., 1989, Cell 59:1107–1113), systems for packaging the much larger rhabdoviral genomic RNAs in vitro are not yet available.
Recently, systems for replication and transcription of DNA-derived minigenomes or small defective RNAs from rhabdoviruses (Conzelmann and Schnell, 1994, J. Virol. 68:713–719; Pattnaik et al., 1992, Cell 69:1011–1120) and paramyxoviruses (Calain et al., 1992, Virology 191:62–71; Collins et al., 1991, Proc. Natl. Acad. Sci. USA 88:9663–9667; Collins et al., 1993, Virology 195:252–256; De and Banerjee, 1993, Virology 196:344–348; Dimock and Collins, 1993, J. Virol. 67:2772–2778; Park et al., 1991, Proc. Natl. Acad. Sci. USA 88:5537–5541) have been described. In these systems, RNAs are assembled into nucleocapsids within cells that express the viral N protein and polymerase proteins. Although these systems have been very useful, they do not allow genetic manipulation of the full-length genome of infectious viruses.
The recovery of rabies virus from a complete cDNA clone was published recently (Schnell et al., 1994, EMBO J. 13:4195–4203). The infectious cycle was initiated by expressing the antigenomic (full-length positive strand) RNA in cells expressing the viral N, P, and L proteins. Although rabies virus is a rhabdovirus, it is structurally and functionally different from the vesiculoviruses. Rabies virus is a Lyssavirus, not a Vesiculovirus. Lyssaviruses invade the central nervous system. Vesiculoviruses invade epithelial cells, predominantly those of the tongue, to produce vesicles. Rabies virus causes encephalitis in a variety of animals and in humans, while VSV causes an epidemic but self-limiting disease in cattle. In sharp contrast to VSV-infected cells, rabies virus produces little or no cytopathic effect in infected cell culture, replicates less efficiently than VSV in cell culture, and causes little depression of cellular DNA, RNA or protein synthesis in infected cell cultures (see Baer et al., 1990, in Virology, 2d ed., Fields et al. (eds.), Raven Press, Ltd., NY, pp. 883, 887). Indeed, there is no cross-hybridization observed between the genomes of rabies virus and VSV, and sequence homology between the two genomes is generally discernable only with the aid of computer run homology programs. The differences between vesiculoviruses and rabies virus, and the extremely rare nature of rabies virus recovery from cDNA (˜108 cells are transfected to yield one infectious cell), renders it unpredictable whether the strategy used with rabies virus would be successful for viruses of a different genus, i.e., the vesiculoviruses.
The recovery of infectious measles virus, another negative strand RNA virus, from cloned cDNA has been attempted, without success (see Ballart et al., 1990, EMBO J. 9(2):379–384 and the retraction thereof by Eschle et al., 1991, EMBO J. 10(11):3558).
2.2. Vaccines
The development of vaccines for the prevention of viral, bacterial, or parasitic diseases is the focus of much research effort.
Traditional ways of preparing vaccines include the use of inactivated or attenuated pathogens. A suitable inactivation of the pathogenic microorganism renders it harmless as a biological agent but does not destroy its immunogenicity. Injection of these “killed” particles into a host will then elicit an immune response capable of preventing a future infection with a live microorganism. However, a major concern in the use of killed vaccines (using inactivated pathogen) is failure to inactivate all the microorganism particles. Even when this is accomplished, since killed pathogens do not multiply in their host, or for other unknown reasons, the immunity achieved is often incomplete, short lived and requires multiple immunizations. Finally, the inactivation process may alter the microorganism's antigens, rendering them less effective as immunogens.
Attenuation refers to the production of strains of pathogenic microorganisms which have essentially lost their disease-producing ability. One way to accomplish this is to subject the microorganism to unusual growth conditions and/or frequent passage in cell culture. Mutants are then selected which have lost virulence but yet are capable of eliciting an immune response. Attenuated pathogens often make good immunogens as they actually replicate in the host cell and elicit long lasting immunity. However, several problems are encountered with the use of live vaccines, the most worrisome being insufficient attenuation and the risk of reversion to virulence.
An alternative to the above methods is the use of subunit vaccines. This involves immunization only with those components which contain the relevant immunological material.
Vaccines are often formulated and inoculated with various adjuvants. The adjuvants aid in attaining a more durable and higher level of immunity using small amounts of antigen or fewer doses than if the immunogen were administered alone. The mechanism of adjuvant action is complex and not completely understood. However, it may involve the stimulation of cytokine production, phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen. Examples of adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), the pluronic polyol L-121, Avridine, and mineral gels such as aluminum hydroxide, aluminum phosphate, etc. Freund's adjuvant is no longer used in vaccine formulations for humans because it contains nonmetabolizable mineral oil and is a potential carcinogen.